Virology 350 (2006) 228–239 www.elsevier.com/locate/yviro

His1 and His2 are distantly related, spindle-shaped haloviruses belonging to the novel group, ⁎ Carolyn Bath 1, Tania Cukalac, Kate Porter, Michael L. Dyall-Smith

Department of Microbiology and Immunology, The University of Melbourne, Parkville, Victoria 3010, Australia Received 7 December 2005; returned to author for revision 31 January 2006; accepted 2 February 2006 Available online 10 March 2006

Abstract

Spindle-shaped are a dominant morphotype in hypersaline waters but their molecular characteristics and their relationship to other archaeal viruses have not been determined. Here, we describe the isolation, characteristics and genome sequence of His2, a spindle-shaped halovirus, and compare it to the previously reported halovirus His1. Their particle dimensions, host-ranges and buoyant densities were found to be similar but they differed in their stabilities to raised temperature, low salinity and chloroform. The genomes of both viruses were linear dsDNA, of similar size (His1, 14,464 bp; His2, 16,067 bp) and mol% G + C (∼40%), with long, inverted terminal repeat sequences. The genomic termini of both viruses are likely to possess bound proteins. They shared little nucleotide similarity and, except for their putative DNA polymerase ORFs, no significant similarity at the predicted protein level. A few of the 35 predicted ORFs of both viruses showed significant matches to sequences in GenBank, and these were always to proteins of haloarchaea. Their DNA polymerases showed 42% aa identity, and belonged to the type B group of replicases that use protein-priming. Purified His2 particles were composed of four main proteins (62, 36, 28 and 21 kDa) and the gene for the major capsid protein was identified. Hypothetical proteins similar to His2 VP1 are present in four haloarchaeal genomes but are not part of complete prophages. This, and other evidence, suggests a high frequency of recombination between haloviruses and their hosts. His1 and His2 are unlike fuselloviruses and have been placed in a new virus group, Salterprovirus. © 2006 Elsevier Inc. All rights reserved.

Keywords: ; Halobacteria; Haloarchaea; Virus; Protein-primed replication; Salt lake

Introduction halovirus isolates (Dyall-Smith et al., 2003; Witte et al., 1997; Zillig et al., 1988). The first spindle-shaped halovirus, His1, was At the extreme salinities found in salt lakes, the majority of reported in 1998 (Bath and Dyall-Smith, 1998) and the first cells are prokaryotes belonging to the archaeal family round halovirus, SH1, was described in 2005 (Porter et al., Halobacteriaceae (Grant et al., 2001). As in marine waters, 2005). However, several years before the His1 report, spindle- the concentration of virus-like particles (VLPs) in salt lakes shaped viruses of Sulfolobus (a thermophilic crenarchaeon) had greatly exceeds that of the cells (Wommack and Colwell, 2000) been discovered and subsequently studied in elegant detail and viruses appear to significantly limit the microbial (Palm et al., 1991; Stedman et al., 2003; Wiedenheft et al., population (Guixa-Boixareu et al., 1996). Direct electron 2004; Zillig et al., 1996). These crenarchaeal viruses were microscopy of hypersaline waters has shown that spindle- classified in a novel family of archaeal viruses, the Fusellovir- shaped and round VLPs are the predominant morphotypes. idae, with the type species being SSV1 (for Sulfolobus spindle- Head-tail particles are less common (Guixa-Boixareu et al., shaped virus 1). 1996; Oren et al., 1997) but represent the majority of reported Since the isolation of SSV1, other spindle-shaped archaeal viruses (and VLPs) with similar properties have been described. These include several viruses of Sulfolobus that ⁎ Corresponding author. Fax: +61 3 9347 1540. E-mail address: [email protected] (M.L. Dyall-Smith). are similar to SSV1 (Prangishvili and Garrett, 2005; Stedman 1 Now at Genetics and Genomics, Primary Industries Research et al., 2003; Wiedenheft et al., 2004); PAV1, a Pyrococcus Victoria, 475 Mickleham Rd., Attwood, Victoria 3049, Australia. VLP (Geslin et al., 2003) that resembles SSV1 in genome

0042-6822/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2006.02.005 C. Bath et al. / Virology 350 (2006) 228–239 229 size and structure (18 kb, circular, dsDNA); and a VLP observed in cultures of Methanococcus voltae A3 (Wood et al., 1989) that has a circular, dsDNA genome (23 kb) and can integrate into the host chromosome (as does SSV1). All of these examples can replicate without obvious lysis of host cultures. The resemblance of His1 to SSV1 in morphology and genome size led us to propose that His1 be included in the family (Bath and Dyall-Smith, 1998) but it was not known at that time if His1 shared a similar replication strategy or any sequence identity with members of this family. Here, we present the isolation and characteristics of a second spindle-shaped halovirus, His2, and show that His1 and His2 are distantly related to each other but are not related to members of the Fuselloviridae or to other spindle-shaped viruses. We also show that their genomes are linear and, probably possess terminal-bound proteins, and are likely to replicate via a protein-primed DNA polymerase. In addition, the major capsid protein of His2 has homologues in three of the five completed haloarchaeal genome sequences.

Results

Isolation, host range and plaque morphology of His2

The Pink Lakes (36° 24.221′ S, 141° 57.556′ E) are a group of relatively small (1.3 km2 or less), closely spaced salt lakes in a protected national park in the remote, semi-arid northwest of Victoria, Australia. In 1995, a water sample collected from these lakes was plated on overlay lawns of hispanica, Haloferax volcanii and Haloferax lucentense. After incubation (2 days, 37 °C), a single plaque formed on the Har. hispanica Fig. 1. Plaques of His1 (A) and His2 (B) on Har. hispanica. Dilutions of virus lawn, and from this a halovirus, designated His2, was isolated were plated with cells in 18% MGM top layer agar, poured on 18% MGM base – that produced clear plaques of about 2 mm diameter, with an plates, allowed to set and incubated at 30 °C (His1) or 37 °C (His2) for 48 72 h for visible plaques. Plates were 8.5 cm in diameter. undefined hazy edge. The plaques were quite distinct from those of His1 (Bath and Dyall-Smith, 1998), a halovirus of Har. hispanica that was isolated in southern Victoria (Fig. 1). The unsuccessful, although uninfected virus-resistant strains, and optimum plaquing temperature for His1 was 30 °C, while His2 carrier-state (persistently infected) strains, could be readily plaqued best at 37 °C. The plaque morphology of His2 was the obtained. Cells of the latter strains did not contain virus DNA in same at both temperatures, but plaques took longer to appear a prophage state, either chromosomally integrated or as a when plates were incubated at 30 °C. (data not shown). The host range of His2 was investigated by spotting a high titred virus lysate (1010 plaque-forming units (PFU) per ml) onto Stability of His2 virions plate overlays containing one of 17 members of the family Halobacteriaceae (see Experimental procedures), including The stability of His2 and His1 under various conditions members of six genera and three additional species of was compared and the results are summarised in Fig. 2.In Haloarcula (Har. marismortui, Har. sinaiiensis and Har. high salt conditions, both viruses maintained their initial vallismortis). Plaques were only observed on lawns of Har. titres for at least 3 weeks when incubated at temperatures hispanica. between 4 and 37 °C (data not shown). At temperatures His2-infected liquid cultures of Har. hispanica produced high above 50 °C, His2 lost infectivity rapidly, while His1 was virus titres (≥1010 PFU/ml). The culture optical density increased slightly more resistant, losing infectivity above 60 °C (Fig. over the first 24 h incubation then decreased to reach a minimum at 2A). A large difference was observed in the stability of His1 2–3 days. Cultures generally remained turbid but, in about 10% of and His2 to low salt: His2 lost infectivity immediately upon cases, complete lysis would occur in small volume cultures (10 ml). dilution while His1 only dropped about twofold in titre over Lysis did not produce higher virus titres. The clear plaques and 1h(Fig. 2B). Both viruses were relatively stable over a occasional lysis of liquid cultures indicated that His2 was lytic in wide pH range (Fig. 2C) and were sensitive to chloroform nature. Attempts to isolate lysogens from infected cultures were (Fig. 2D). Download English Version: https://daneshyari.com/en/article/3427597

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